Luc pair mir luciferase assay

Dual-reporter expression clones of murine wild-type and mutated PGC1-α 3′-UTR were obtained from Genecopoeia (Rockville, MD, USA). The sequences are shown in Fig. 4A. The mutant PGC1-α 3′-UTR reporter was created by mutating the seed regions of the predicted mmu-miR-494-3p site (AUGUUUC to CUGCUCC). The 3′-UTR sequences were inserted in the pEZX-MT06 vector (Genecopoeia) downstream of a firefly luciferase reporter gene driven by an SV40 enhancer. The Renilla Luciferase reporter was used as an internal control for transfection efficiency. The plasmids were transfected into beige-type 3T3-L1 cells using DharmaFECT Duo (Cat# T-2010-03; Dharmacon, Lafayette, CO, USA) according to the manufacturer’s instructions. Dual luciferase activity was measured using the Luc-Pair miR luciferase assay (Genecopoeia) according to the manufacturer’s instructions.

HEK293 cells were plated in 96 well plates (3.0–4.0 × 10⁴) in antibiotic-free complete growth media (EMEM + 10% FBS) 24 h prior to transfection. Transfections (three separate experiments) were performed in triplicate by adding 10 ul of transfection complex containing 0.5 ul Lipofectamine 2000 (Invitrogen), 100 ng of Bdnf miRNA Target Sequence 3′UTR expression clone (GeneCopoeia), 60 nmol/L miR-15b mimic (Dharmacon), or 37 nmol/L antisense LNA oligo (Exiqon). Experimental conditions included plasmid alone, plasmid + miR-15b mimic, and plasmid + miR-15b mimic and LNA inhibitor. Wells with cells alone were used to subtract background expression when read on the plate reader. Then, 24 h after transfection cells, were lysed and both Firefly luciferase and Renilla luciferase expression was measured using the Luc-Pair miR Luciferase Assay (GeneCopoeia) in a GloMax^®-Multi+ Detection System with Instinct™ Software (Promega). After background subtraction, Firefly luminescence was normalized to Renilla luminescence producing relative luciferase unit (RLU) values. Data are reported as a percent change from the BDNF plasmid alone levels, displayed with 95% confidence intervals. A paired t-test was used to compare the miR-15b mimic groups and miR-15b mimic plus inhibitor groups to BDNF plasmid alone groups. All calculations were done in R.

Firefly luciferase activity was measured at room temperature with the Luc-Pair miR Luciferase Assay Kit (GeneCopoeia) using a single-tube luminometer (Model TD 20/20 Turner Designs, Sunnyvale, CA). Dual luciferase reporter constructs containing the 3′- untranslated region (UTR) of the PDIA1 gene with miR-204-binding sites 5′-(AAAGGGA)-3′ (GeneCopoeia) or 3′-UTR of PDIA1 with mutated miR-204-binding site 5′-(TCGATTC)-3′ (custom cloned at Emory Molecular Biology Core Facility) were transfected into human umbilical vein endothelial cells (HUVECs) using a Nucleofector Kit (Lonza). HUVECs were transfected first with wild-type or mutated target gene 3′-UTR using the HUVEC Nucleofector Kit (Lonza) and were allowed to recover for 24 h. The second transient transfection was performed using increasing concentrations of miR-mimic-204 or respective mismatch controls using Oligofectamine 3000 (Invitrogen). Firefly and Renilla luciferase activities were measured using a Luc-Pair miR Luciferase Assay (GeneCopoeia) as per the manufacturer’s recommendations.

Transient transfections of HeLa cells were performed by using Lipofectamine 2000 (Invitrogen) according to the manufacturer’s instructions. For the luciferase assay, cells were plated at a density of 10⁵ cells/well in 24-well plates and co-transfected with either 100 ng of MiTarget MicroRNA 3′ UTR Target Clone HmiT019538-MT01 (GeneCopoeia, NR4A3 3′ UTR) or HmiT017632-MT01 (GeneCopoeia, SOX5 3′ UTR), as well as 20 nM miRNA mimics (hsa-miR-96-5p for SOX5 3′ UTR, and hsa-miR-7-5p, and hsa-miR-17-5p for NR4A3 3′ UTR, respectively) per well. Control wells were transfected with either HmiT019538-MT01 or HmiT017632-MT01 plasmid, and allstars negative-control siRNA AF 488 (Qiagen). Transfection efficiency was found to be 100%. HmiT019538-MT0 and HmiT017632-MT01 plasmids express both firefly and renilla luciferase. Firefly and renilla luciferase activities were measured 24 hours after transfection by using the Luc-Pair miR luciferase assay (GeneCopoeia) and a GloMax 96 Microplate Luminometer (Promega). Firefly luciferase activity was normalized to renilla luciferase activity for each transfected well. In all the experiments, transfection and luciferase assays were performed in triplicate.

The miRNA 3′UTR target clone (Luc-MET-3′UTR) was purchased from GeneCopoeia (Rockville, MD), which contains renilla luciferase as internal control fused downstream to a firefly luciferase. The cells were co-transfected with control or c-Met 3′UTR luciferase vector and miR-122 mimic (or inhibitor) or its relative control using FuGENE^® HD Reagent (Roche, Indianapolis, IN). After 48 h of transfection, firefly and renilla luciferase activities were measured using Luc-Pair miR Luciferase Assay (GeneCopoeia) according to the manufacturer's protocols.

PANC1, MDA-MB-231, and HEK293 normal epithelial cells were transfected with pEZX-MT06 miRNA reporter vectors containing the binding sites for miR-873 in the 3′ UTR of KRAS and the luciferase gene (GeneCopoeia, Rockville, MD). Cells were plated (5 × 10⁴ cells per well) in each well of a 24-well plate 24 h before transfection. The cells were transfected with the pEZX-MT06 vector (200 ng) and 50 nM miR-873 mimic or control miRNA. Luciferase activity was measured 48 h after transfection using the Luc-Pair miR Luciferase Assay (GeneCopoeia). For each sample, firefly luciferase activity was normalized to Renilla luciferase activity.